技术资料

Cultures of human embryonic stem cell typically rely on protein matrices or feeder cells to support attachment and growth,while mechanical,enzymatic or chemical cell dissociation methods are used for cellular passaging. However,these methods are ill defined,thus introducing variability into the system,and may damage cells. They also exert selective pressures favouring cell aneuploidy and loss of differentiation potential. Here we report the identification of a family of chemically defined thermoresponsive synthetic hydrogels based on 2-(diethylamino)ethyl acrylate,which support long-term human embryonic stem cell growth and pluripotency over a period of 2-6 months. The hydrogels permitted gentle,reagent-free cell passaging by virtue of transient modulation of the ambient temperature from 37 to 15 °C for 30 min. These chemically defined alternatives to currently used,undefined biological substrates represent a flexible and scalable approach for improving the definition,efficacy and safety of human embryonic stem cell culture systems for research,industrial and clinical applications. View Publication

Many protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function,little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet,understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore,we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs,from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties,including resting membrane potential,action potential,sodium and potassium channel currents,somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons,the resting membrane potential became more negative,the expression of voltage-gated sodium channels increased,the membrane became capable of generating action potentials following adequate depolarization and,at day 48-55,50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step,of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca2+ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology,as electrophysiological properties of iPSC-derived neurons mature over time. View Publication

Manipulation of the CD28/CTLA-4 pathway is at the heart of a number of immunomodulatory approaches used in both autoimmunity and cancer. Although it is clear that CTLA-4 is a critical regulator of T cell responses,the immunological contexts in which CTLA-4 controls immune responses are not well defined. In this study,we show that whereas CD80/CD86-dependent activation of resting human T cells caused extensive T cell proliferation and robust CTLA-4 expression,in this context CTLA-4 blocking Abs had no impact on the response. In contrast,in settings where CTLA-4(+) cells were present as regulators� View Publication

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development,however,is restricted by significant off-tumor toxicities associated with Fc$\gamma$R interactions. Here,we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody,1D8N/CEGa1,consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly,treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate Fc$\gamma$R interactions in the 4-1BB-agonist-associated immune abnormalities,and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy. View Publication

Glioblastoma growth is driven by cancer cells that have stem cell properties,but molecular determinants of their tumorigenic behavior are poorly defined. In cancer,altered activity of the epigenetic modifiers Polycomb and Trithorax complexes may contribute to the neoplastic phenotype. Here,we provide the first mechanistic insights into the role of the Trithorax protein mixed lineage leukemia (MLL) in maintaining cancer stem cell characteristics in human glioblastoma. We found that MLL directly activates the Homeobox gene HOXA10. In turn,HOXA10 activates a downstream Homeobox network and other genes previously characterized for their role in tumorigenesis. The MLL-Homeobox axis we identified significantly contributes to the tumorigenic potential of glioblastoma stem cells. Our studies suggest a role for MLL in contributing to the epigenetic heterogeneity between tumor-initiating and non-tumor-initiating cells in glioblastoma. View Publication

Cell-fate transitions involve the integration of genomic information encoded by regulatory elements,such as enhancers,with the cellular environment. However,identification of genomic sequences that control human embryonic development represents a formidable challenge. Here we show that in human embryonic stem cells (hESCs),unique chromatin signatures identify two distinct classes of genomic elements,both of which are marked by the presence of chromatin regulators p300 and BRG1,monomethylation of histone H3 at lysine 4 (H3K4me1),and low nucleosomal density. In addition,elements of the first class are distinguished by the acetylation of histone H3 at lysine 27 (H3K27ac),overlap with previously characterized hESC enhancers,and are located proximally to genes expressed in hESCs and the epiblast. In contrast,elements of the second class,which we term 'poised enhancers',are distinguished by the absence of H3K27ac,enrichment of histone H3 lysine 27 trimethylation (H3K27me3),and are linked to genes inactive in hESCs and instead are involved in orchestrating early steps in embryogenesis,such as gastrulation,mesoderm formation and neurulation. Consistent with the poised identity,during differentiation of hESCs to neuroepithelium,a neuroectoderm-specific subset of poised enhancers acquires a chromatin signature associated with active enhancers. When assayed in zebrafish embryos,poised enhancers are able to direct cell-type and stage-specific expression characteristic of their proximal developmental gene,even in the absence of sequence conservation in the fish genome. Our data demonstrate that early developmental enhancers are epigenetically pre-marked in hESCs and indicate an unappreciated role of H3K27me3 at distal regulatory elements. Moreover,the wealth of new regulatory sequences identified here provides an invaluable resource for studies and isolation of transient,rare cell populations representing early stages of human embryogenesis. View Publication

Most cancer vaccines target peptide antigens,necessitating personalization owing to the vast inter-individual diversity in major histocompatibility complex (MHC) molecules that present peptides to T cells. Furthermore,tumours frequently escape T cell-mediated immunity through mechanisms that interfere with peptide presentation1. Here we report a cancer vaccine that induces a coordinated attack by diverse T cell and natural killer (NK) cell populations. The vaccine targets the MICA and MICB (MICA/B) stress proteins expressed by many human cancers as a result of DNA damage2. MICA/B serve as ligands for the activating NKG2D receptor on T cells and NK cells,but tumours evade immune recognition by proteolytic MICA/B cleavage3,4. Vaccine-induced antibodies increase the density of MICA/B proteins on the surface of tumour cells by inhibiting proteolytic shedding,enhance presentation of tumour antigens by dendritic cells to T cells and augment the cytotoxic function of NK cells. Notably,this vaccine maintains efficacy against MHC class I-deficient tumours resistant to cytotoxic T cells through the coordinated action of NK cells and CD4+ T cells. The vaccine is also efficacious in a clinically important setting: immunization following surgical removal of primary,highly metastatic tumours inhibits the later outgrowth of metastases. This vaccine design enables protective immunity even against tumours with common escape mutations. View Publication

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations,including their reliance on exogenous materials or electrical fields,which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30–80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material,such as carbon nanotubes,proteins,and siRNA,to 11 cell types,including embryonic stem cells and immune cells. When used for the delivery of transcription factors,the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed,its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications. View Publication

BackgroundRecent studies highlight the critical role of microglia in neurodegenerative disorders,and emphasize the need for humanized models to accurately study microglial responses. Human-mouse microglia xenotransplantation models are a valuable platform for functional studies and for testing therapeutic approaches,yet currently those models are only available for academic research. This hampers their implementation for the development and testing of medication that targets human microglia.MethodsWe developed the hCSF1Bdes mouse line,which is suitable as a new transplantation model and available to be crossed to any disease model of interest. The hCSF1Bdes model created by CRISPR gene editing is RAG2 deficient and expresses human CSF1. Additionally,we crossed this model with two humanized App KI mice,the AppHu and the AppSAA. Flow cytometry,immunohistochemistry and bulk sequencing was used to study the response of microglia in the context of Alzheimer’s disease.ResultsOur results demonstrate the successful transplantation of iPSC-derived human microglia into the brains of hCSF1Bdes mice without triggering a NK-driven immune response. Furthermore,we confirmed the multipronged response of microglia in the context of Alzheimer’s disease. The hCSF1Bdes and the crosses with the Alzheimer’s disease knock-in model AppSAA and the humanized App knock-in control mice,AppHu are deposited with EMMA and fully accessible to the research community.ConclusionThe hCSF1Bdes mouse is available for both non-profit and for-profit organisations,facilitating the use of the xenotransplantation paradigm for human microglia to study complex human disease.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13024-025-00823-2. View Publication

A fundamental impediment to understanding the brain is the availability of inexpensive and robust methods for targeting and manipulating specific neuronal populations. The need to overcome this barrier is pressing because there are considerable anatomical,physiological,cognitive and behavioral differences between mice and higher mammalian species in which it is difficult to specifically target and manipulate genetically defined functional cell types. In particular,it is unclear the degree to which insights from mouse models can shed light on the neural mechanisms that mediate cognitive functions in higher species,including humans. Here we describe a novel recombinant adeno-associated virus that restricts gene expression to GABAergic interneurons within the telencephalon. We demonstrate that the viral expression is specific and robust,allowing for morphological visualization,activity monitoring and functional manipulation of interneurons in both mice and non-genetically tractable species,thus opening the possibility to study GABAergic function in virtually any vertebrate species. View Publication